Comparison of 6 DNA extraction methods for isolation of high yield of high molecular weight DNA suitable for shotgun metagenomics Nanopore sequencing to detect bacteria

BMC Genomics

Research

Nancy H. C. Roosens¹  Bram Bloemen¹  Sigrid C. J. De Keersmaecker¹  Kevin Vanneste¹  Mathieu Gand¹ 

[1] Transversal Activities in Applied Genomics, Sciensano, Rue Juliette Wytsman 14, 1050, Brussels, Belgium;

关键词: DNA extraction;    Method evaluation;    HMW DNA;    Nanopore;    Metagenomics;    Long-read;   

DOI  :  10.1186/s12864-023-09537-5

 received in 2023-04-18, accepted in 2023-07-27,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundOxford Nanopore Technologies (ONT) offers an accessible platform for long-read sequencing, which improves the reconstruction of genomes and helps to resolve complex genomic contexts, especially in the case of metagenome analysis. To take the best advantage of long-read sequencing, DNA extraction methods must be able to isolate pure high molecular weight (HMW) DNA from complex metagenomics samples, without introducing any bias. New methods released on the market, and protocols developed at the research level, were specifically designed for this application and need to be assessed.ResultsIn this study, with different bacterial cocktail mixes, analyzed as pure or spiked in a synthetic fecal matrix, we evaluated the performances of 6 DNA extraction methods using various cells lysis and purification techniques, from quick and easy, to more time-consuming and gentle protocols, including a portable method for on-site application. In addition to the comparison of the quality, quantity and purity of the extracted DNA, the performance obtained when doing Nanopore sequencing on a MinION flow cell was also tested. From the obtained results, the Quick-DNA HMW MagBead Kit (Zymo Research) was selected as producing the best yield of pure HMW DNA. Furthermore, this kit allowed an accurate detection, by Nanopore sequencing, of almost all the bacterial species present in a complex mock community.ConclusionAmongst the 6 tested methods, the Quick-DNA HMW MagBead Kit (Zymo Research) was considered as the most suitable for Nanopore sequencing and would be recommended for bacterial metagenomics studies using this technology.

【 授权许可】

CC BY   
© BioMed Central Ltd., part of Springer Nature 2023

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